The membrane proximal region (MPR, residues 649–683) and transmembrane domain (TMD, residues 684–705) of the gp41 subunit of HIV-1’s envelope protein are highly conserved and are important in viral mucosal transmission, virus attachment and membrane fusion with target cells. Several structures of the trimeric membrane proximal external region (residues 662–683) of MPR have been reported at the atomic level; however, the atomic structure of the TMD still remains unknown. To elucidate the structure of both MPR and TMD, we expressed the region spanning both domains, MPR-TM (residues 649–705), in Escherichia coli as a fusion protein with maltose binding protein (MBP). MPR-TM was initially fused to the C-terminus of MBP via a 42 aa-long linker containing a TEV protease recognition site (MBP-linker-MPR-TM).
Biophysical characterization indicated that the purified MBP-linker-MPR-TM protein was a monodisperse and stable candidate for crystallization. However, crystals of the MBP-linker-MPR-TM protein could not be obtained in extensive crystallization screens. It is possible that the 42 residue-long linker between MBP and MPR-TM was interfering with crystal formation. To test this hypothesis, the 42 residue-long linker was replaced with three alanine residues. The fusion protein, MBP-AAA-MPR-TM, was similarly purified and characterized. Significantly, both the MBP-linker-MPR-TM and MBP-AAA-MPR-TM proteins strongly interacted with broadly neutralizing monoclonal antibodies 2F5 and 4E10. With epitopes accessible to the broadly neutralizing antibodies, these MBP/MPR-TM recombinant proteins may be in immunologically relevant conformations that mimic a pre-hairpin intermediate of gp41.
Viral protein U (Vpu) is a type-III integral membrane protein encoded by Human Immunodeficiency Virus-1 (HIV- 1). It is expressed in infected host cells and plays several roles in viral progeny escape from infected cells, including down-regulation of CD4 receptors. But key structure/function questions remain regarding the mechanisms by which the Vpu protein contributes to HIV-1 pathogenesis. Here we describe expression of Vpu in bacteria, its purification and characterization. We report the successful expression of PelB-Vpu in Escherichia coli using the leader peptide pectate lyase B (PelB) from Erwinia carotovora. The protein was detergent extractable and could be isolated in a very pure form. We demonstrate that the PelB signal peptide successfully targets Vpu to the cell membranes and inserts it as a type I membrane protein. PelB-Vpu was biophysically characterized by circular dichroism and dynamic light scattering experiments and was shown to be an excellent candidate for elucidating structural models.
Limited to streaming only those videos a vendor hosted, ASU Libraries sought to expand collection options with a trial project for hosting content locally. Kaltura, was selected as the platform, but Kaltura does not work out of the box. This presentation will cover how using Drupal, along with Kaltura, we built a working video hosting solution. The presentation will cover administrative hurdles, stumbling blocks, pitfalls, enhancements, and lessons learned along the way.
You’ve probably heard a lot of “futurists” talk about data, but it’s not always clear how data relate to our day to day work in libraries.
Why are data important, and what’s the big deal? Data are not just spreadsheets and numbers, but come in many different shapes, colors, and flavors! In this presentation, we will give an introduction to data, talk about why it is relevant, and demonstrate how to and use data in practical situations. We will also provide innovative examples that will inspire you to connect with your colleagues and patrons!
The Arizona State University Libraries’ fun Library Minute video series brings information about resource and services to a large student body. For the first time, we present a workshop walking through the entire production process from start to finish and offering suggestions on how to fit multimedia into your marketing and outreach strategy. In this session, we will produce a short video with participants in three steps:
1. Conceptualization and Planning.
2. Recording.
3. Editing and Distribution.
Digital Production Manger Matthew Harp will demonstrate the tools and process and elaborate on the use of social media, YouTube, and the Internet Archive in the distribution plan. Together with Mimmo Bonanni and Library Minute Host Anali Perry, we’ll share our tips and tricks for video production using whatever resources are available.
Presented at the 2011 Arizona Library Association Conference 2011 - Tucson, Arizona